How to survive pig farming: Mechanism of SCCmec element deletion and metabolic stress adaptation in livestock-associated MRSA

Previous research on methicillin susceptible Staphylococcus aureus (MSSA) belonging to livestock-associated (LA-) sequence type (ST) 398, isolated from pigs and their local surroundings, indicated that differences between these MSSA and their methicillin resistant predecessors (MRSA) are often limited to the absence of the staphylococcal cassette chromosome mec (SCCmec) and few single nucleotide polymorphisms. So far, our understanding on how LA-MRSA endure the environmental conditions associated with pig-farming as well as the putative impact of this particular environment on the mobilisation of SCCmec elements is limited. Thus, we performed in-depth genomic and transcriptomic analyses using the LA-MRSA ST398 strain IMT38951 and its methicillin susceptible descendant. We identified a mosaic-structured SCCmec region including a putative replicative SCCmecVc which is absent from the MSSA chromosome through homologous recombination. Based on our data, such events occur between short repetitive sequences identified within and adjacent to two distinct alleles of the large cassette recombinase genes C (ccrC). We further evaluated the global transcriptomic response of MRSA ST398 to particular pig-farm associated conditions, i.e., contact with host proteins (porcine serum) and a high ammonia concentration. Differential expression of global regulators involved in stress response control were identified, i.e., ammonia-induced alternative sigma factor B-depending activation of genes for the alkaline shock protein 23, the heat shock response and the accessory gene regulator (agr)-controlled transcription of virulence factors. Exposure to serum transiently induced the transcription of distinct virulence factor encoding genes. Transcription of genes reported for mediating the loss of methicillin resistance, especially ccrC, was not significantly different compared to the unchallenged controls. We concluded that, from an evolutionary perspective, bacteria may save energy by incidentally dismissing a fully replicative SCCmec element in contrast to the induction of ccr genes on a population scale. Since the genomic SCCmec integration site is a hot-spot of recombination, occasional losses of elements of 16 kb size may restore capacities for the uptake of foreign genetic material. Subsequent spread of resistance, on the other hand, might depend on the autonomous replication machinery of the deleted SCCmec elements that probably enhance chances for reintegration of SCCmec into susceptible genomes by mere multiplication.

Previous research on methicillin susceptible Staphylococcus aureus (MSSA) belonging to livestock-associated (LA-) sequence type (ST) 398, isolated from pigs and their local surroundings, indicated that differences between these MSSA and their methicillin resistant predecessors (MRSA) are often limited to the absence of the staphylococcal cassette chromosome mec (SCCmec) and few single nucleotide polymorphisms. So far, our understanding on how LA-MRSA endure the environmental conditions associated with pig-farming as well as the putative impact of this particular environment on the mobilisation of SCCmec elements is limited. Thus, we performed in-depth genomic and transcriptomic analyses using the LA-MRSA ST398 strain IMT38951 and its methicillin susceptible descendant. We identified a mosaic-structured SCCmec region including a putative replicative SCCmecVc which is absent from the MSSA chromosome through homologous recombination. Based on our data, such events occur between short repetitive sequences identified within and adjacent to two distinct alleles of the large cassette recombinase genes C (ccrC). We further evaluated the global transcriptomic response of MRSA ST398 to particular pig-farm associated conditions, i.e., contact with host proteins (porcine serum) and a high ammonia concentration. Differential expression of global regulators involved in stress response control were identified, i.e., ammonia-induced alternative sigma factor B-depending activation of genes for the alkaline shock protein 23, the heat shock response and the accessory gene regulator (agr)-controlled transcription of virulence factors. Exposure to serum transiently induced the transcription of distinct virulence factor encoding genes. Transcription of genes reported for mediating the loss of methicillin resistance, especially ccrC, was not significantly

Introduction
Since the mid-2000s, sequence type (ST)398 and its closely related descendants represent the predominant clonal complex (CC) of livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) throughout Europe (Butaye et al., 2016). ST398 LA-MRSA most likely originate from a lineage of human methicillin susceptible S. aureus (MSSA) and have since adapted to livestock (particularly to pig farming) via acquisition of resistance genes against tetracycline and beta-lactam antibiotics (Price et al., 2012). ST398 LA-MRSA are also capable of causing infections in humans (Williamson et al., 2014;Sieber et al., 2019;Lu et al., 2021), a problem that is of particular concern across regions with high pig farming density (Köck et al., 2009;Vanderhaeghen et al., 2010;Gómez et al., 2020). Indeed, some MRSA are prone to cross species barriers (Sheppard et al., 2018) and are capable of rapid adaptation to different habitats and environmental conditions (Larsen et al., 2022). Adaptation processes were mainly studied on the genomic level and include either changes of the core genome (Huber et al., 2020) or an uptake of mobile genetic elements (MGEs; Mccarthy and Lindsay, 2013;Walther et al., 2018;Sieber et al., 2019;Huber et al., 2020;Leinweber et al., 2021).
Genome alterations also affect the staphylococcal cassette chromosome mec (SCCmec) including its mec complex which confers broad-spectrum β-lactam resistance in S. aureus and whose spontaneous loss was reported for ST398 isolates (Chlebowicz et al., 2010;Gómez et al., 2020). SCCmec elements represent large genomic islands of various sizes and compositions, which are inserted into the highly conserved region between the rlmH (previously orfX) and dus (previously orfY) genes of staphylococcal chromosomes (Semmler et al., 2016). Loss of SCCmec elements can either result from homologous recombination (HR) events (involving the recombination apparatus of the cell) or through precise excision of the element from its integration site. The latter process is driven by the SCCmec-encoded site-specific serine recombinases (i.e., ccrA/B or ccrC) which also mediate integration of the element into the rlmH gene (Hanssen and Ericson Sollid, 2006;Chlebowicz et al., 2010;Wang and Archer, 2010;Liu et al., 2017;Gómez et al., 2020).
Interestingly, loss of SCCmec is thought to provide an advantage for S. aureus in challenging environments, for example when exposed to antibiotics (Noto et al., 2008) and/or the host immune system (Read et al., 2018;Tickler et al., 2020); and in LA-MRSA, the specific environmental conditions surrounding pig farming were proposed as factors that might facilitate deletion of the element (Nübel et al., 2010;Vandendriessche et al., 2014).
In industrial pig farming, the toxic gas ammonium hydroxide (NH 3 ) is released from manure during (bacterial) deamination of proteins fed to the animals. The gas subsequently accumulates in the air of the barn and, due to its high solubility in fluids, it is harmful to airway mucosal surfaces of pig farm workers and exposed pigs (Urbain et al., 1996;Wang et al., 2020) Thus, it is unavoidable that MRSA colonizing moist mucosal surfaces of the anterior nostrils of pigs are exposed to increased concentrations of ammonia. Therefore, exposure to high concentrations of ammonia is a condition commonly encountered by LA-MRSA ST398, and a recent study demonstrated that these bacteria indeed survive under such conditions (Astrup et al., 2021).
Regarding cytosolic pH, ammonia is the preferred nitrogen source of most microorganisms, and once in the cell, is directed to glutamine synthesis (Gruswitz et al., 2007). However, information on ammonia-induced (metabolic) gene expression patterns in LA-MRSA are currently scarce. Aside from exposure to the harsh barn environment, LA-MRSA remain associated with pigs as their primary host, which illustrates another condition the bacteria are required to adapt to.
Taking these different environmental conditions into account, the aims of this study were: (i) to reconstruct the region downstream of rlmH in an ST398 MRSA and its methicillin susceptible descendant (MSSA) in order to reveal the mechanisms involved in the loss of SCCmec; (ii) to evaluate the global transcriptomic response of MRSA ST398 exposed to particular LA environmental conditions, i.e., contact with host proteins (porcine Frontiers in Microbiology 03 frontiersin.org serum) and enhanced ammonia concentrations, with a special focus on (iii) transcription levels of genes previously reported as being associated with the acquisition or loss of SCCmec elements.

DNA isolation, whole-genome sequencing and assembly
Genomic bacterial DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, Venlo, Netherlands) according to the manufacturer's instructions. Isolates were pre-incubated with 0.1 mg/ml Lysostaphin and 0.1 mg/ml Proteinase K (both from Sigma-Aldrich, St. Louis, Missouri, United States). NanoDrop Spectrophotometer (Thermo Fisher Scientific, United States) as well as the Qubit 2.0 Fluorometer (Invitrogen, United States) were used to evaluate the DNA quality and quantity, respectively.
Whole-genome sequencing (WGS) was performed using an Illumina MiSeq which resulted in 300 bp paired-end reads with an obtained coverage >90X. Furthermore, size selection was conducted on the DNA using a 10 K Blue Pippin kit for additional sequencing with the Single Molecule Real-Time (SMRT) Sequencing Technology on a PacBio RS II by GATC Biotech AG (Konstanz, Germany). PacBio raw data was assembled by Flye v2.8.2-b1689 (Kolmogorov et al., 2019). Adapter-trimmed Illumina short reads were used for assembly correction utilizing the polishing procedure of unicycler v0.4.8 (Wick et al., 2017). De novo assembled genomes were annotated using Prokka v1.14.6 (Seemann, 2014). Both genomes were comparatively investigated with respect to single nucleotide polymorphisms (SNPs), insertions and deletions by read mapping utilizing botwie2 v2.3.0 (Langmead and Salzberg, 2012). Putative sequence variants were defined as divergent nucleotides with a minimum coverage of 10X and an allele frequency of >80%. ResFinder-4.1 (Zankari et al., 2012;Bortolaia et al., 2020) was used to identify resistance genes, and further genomic sites of interest were investigated using Geneious 11.1.5 (Biomatters Ltd., Australia; Kearse et al., 2012). PHASTER (Arndt et al., 2016) was utilized to identify putative (pro-) phages across both genomes.

Selection of RNA Seq samples
Cells of IMT38951 were grown in 60 ml MH II and incubated overnight at 37°C and 250 rpm and, on the following day, diluted to an OD 600 of 0.1 in 100 ml of fresh MH II and further incubated at 37°C and 250 rpm.
Based on previous research (Anderson et al., 2010;Malachowa et al., 2011;Cardile et al., 2014;Pinon et al., 2015) we used an ammonia concentration of 0.3% (Th. Geyer, Renningen, Germany), 10% porcine serum (Innovative Research Inc., Peary Court Novi, Michigan, United States) as well as a combination of both in order to mimic environmental conditions associated with pig-farming. Sampling time points were selected based on results of Stojanov et al. (2015) on SCCmec excision and the major regulative activity of the accessory gene regulator (agr)-system (Wang and Muir, 2016;Huber et al., 2020). At an OD 600 of 0.4, the broths were supplemented with either porcine serum (10%), ammonia (0.3%), or both. To ensure the viability of the bacteria in the supplemented media, the viable plate count was determined every 15 min (0-90 min after media supplementation). Media from controls were not supplemented.
A previously published protocol (Lavoie and Summers, 2018) was used to select the samples for RNA sequencing to guarantee overall comparability. Briefly, for each of the experiments, seven biological replicates were initially grown alongside seven corresponding controls. The OD 600 was determined for each set of samples and controls (Supplementary Table 1). Then, three out of the seven samples that exhibited the most similar OD 600 values per growth condition and time point (10 and 60 min after media supplementation) were selected for subsequent RNA isolation (n = 18 samples). In addition, the three most similar control samples per growth condition and time point were selected as well (n = 18; written in bold in Supplementary Table 1). Each aliquot was centrifuged at 20,000 g and 4°C for 3 min. The supernatant was aspirated, and the pellet was immediately frozen at −80°C following previously published protocols (Mäder et al., 2016;Lavoie and Summers, 2018).

RNA sequencing and differential transcriptomic analysis
In total, 36 cell pellets (18 samples and 18 associated controls) were shipped to LGC Genomics GmbH (Berlin, Germany), where the RNA was isolated using the RNASnap method (Stead et al., 2012). RNA sequencing was performed on an Illumina NextSeq 500/550 V2, resulting in one channel paired-end reads. Details of the company's standard protocols for quality control, RNA extraction and rRNA depletion using Ribo-Zero (Epicentre  (Wolf et al., 2020) was used to perform differential RNA sequencing analysis by comparing the transcriptomic profiles of selected challenges (ammonia, porcine serum or the combination of them) with control samples, resulting in the identification of differentially expressed genes (DEGs) across the sample set. Initial analysis steps including read preprocessing (trimming of low-quality ends with Phred score < 20), mapping to the genomic reference, transcript quantification (genes below 10 counts were discarded), statistical identification of differentially expressed genes between sample groups (corrected p ≤ 0.05), overrepresentation analysis of associated Gene Ontologies (GO) and visualization of affected Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed within SCORE. An additional fold change filter (log2FC > 1) was applied to ensure biological relevancy of the DEGs (Slany et al., 2017). Genes not fulfilling these criteria were counted as not being differentially expressed between conditions. The resulting expression tables were subsequently merged and additionally characterized using CD-Search v3.18 (Marchler-Bauer and Bryant, 2004) and eggNOG-mapper v2.0.5 (Huerta-Cepas et al., 2019). Principal component analysis (PCA) was performed based on normalized transcripts per million (TPM) expression values to assess intersample distances (Scholes and Lewis, 2020).

Results
Reconstruction of the SCCmec integration site downstream of rlmH revealed the loss of a complete SCCmecVc element harbouring a replication machinery To allow determination of the precise genomic sites flanking the loss of a mecA-containing sequence in MSSA IMT38951_42, a finished whole-genome reference sequence was required. PacBio long reads were utilized to assemble the draft genomes using Flye, resulting in a coverage of >335-times and > 331-times. Short-read polishing of the genomes was performed, providing an additional 161X and 187X sequence coverage to the reconstructed genomes of IMT38951 (MRSA) and IMT38951_42, respectively ( Figure 1).
Pairwise distance and relatedness ( Figure 1) was calculated, revealing a total difference of 10 SNPs (excluding phage regions) and the lack of a 16 kb sequence downstream of the SCCmec integration site (rlmH) in IMT38951_42 ( Figure 2). A closer inspection of the region revealed the loss of a complete SCCmecVc element ( Figure 2) comprising the ccrC8 type recombinase, the C2 mec complex and a recently described putative operon containing an A-family DNA polymerase (CCPol) together with a small protein lacking conserved domains ("middle protein, " MP) as well as a putative helicase (Cch2; referred to as "CCPol-MP-Cch2" I & II in Figures 2, 3), as recently described by Bebel et al. (2020). The region downstream of rlmH in IMT38951 includes a notable repertoire of homologues sequences, i.e., two distinct but closely related ccrC variants and two nearly homologues intergenic spacer regions of 224 bp length, four transposases (tnp) of insertion sequence (IS) element 431 (IS431) and two variants of the replication machinery CCPol-MP-Cch2 ( Figure  2; Supplementary Table 2) (region rlmH -dus, highlighted in green). The data suggest that the deletion of SCCmecVc occurred during HR between either ccrC8 and ccrC1 (first 48 bp are 100% identical) or the terminal 38 bp (identity: 100%) of the two 224 bp-comprising intergenic spacer regions upstream of the ccrC variants, respectively ( Figure 3) that provide at least two distinct possibilities for HR without any further sequence alterations apart from the loss of the SCCmec element within this region. Both intergenic spacer sequences present in the MRSA variant harbor the original (identical) promoter region of ccrC variants, including the 19 bp SOS box described by Liu et al. (Liu et al., 2017;Supplementary Figure 1). Interestingly, the deleted SCCmecVc element still harbours its putative autonomous replication apparatus, since all loci involved (direct repeats, inverted repeats, origin of replication, CCPol-MP-Cch2 II) remain completely unaffected by the proposed recombination event (Supplementary Figure 1).
A ccr-mediated integration and excision, on the other hand, is known to change the number of 15 bp DR in the region downstream of rlmH (Semmler et al., 2016). Here, the total number and location of the two distinct 15 bp repeats present in Further sequence comparison with the type V SCCmec (GenBank: AB512767) genes and intergenic regions of a strain (TSGH17) belonging to another livestock-associated genetic Genome-wide comparison of MRSA ST398 (IMT38951) and its descendant MSSA (IMT38951_42). A combinatorial approach including long reads (PacBio) and short reads (Illumina sequencing) allowed precise reconstruction of both genomes. Five regions harbouring phages or phageassociated genes identified using PHASTER are indicated in green. Location of resistance genes identified using ResFinder 2.1 and the SCCmec intergration site are indicated in blue. Comparative genomics determined a difference of 10 single nucleotide polymorphisms in total (excluding phage regions). Sequence comparison of IMT38951 and IMT38951_42 from rlmH (previously: orfX) to dus (previously orfY). Repetitive elements and homologues regions challenged the precise reconstruction of MRSA IMT38951 and its isogenic descendant lacking SCCmecVc (region B). Since sequence identities of genes and intergenic spacer regions present in this region range from 98/99% (light blue-green) to 100% (blue-green), including sequences and genes that flank (regions A and C) the missing SCCmec element in IMT38951_42. DR, direct repeat of 15 bp length; CCPol-MP-Cch2, operon containing an A-family DNA polymerase (CCPol) together with a small protein lacking conserved domains ("middle protein," MP) as well as a putative helicase (Cch2) (Bebel et al., 2020); ccrC, chromosomal cassette recombinase C; tpn, transposase associated with insertion element (IS) 431; ISR, intergenic spacer region of 224 bp length. Exposure of ST398 to porcine serum and ammonia alters activity of regulatory pathways and genes involved in environmental adaptation The transcriptomic profiles of IMT38951 triplicate samples challenged by environment-mimicking conditions such as an increased ammonia concentration (pH 9.0), porcine serum (pH 7.3) and a combination of them (pH 8.9) were compared to corresponding unchallenged control samples (pH 7.2) after 10 and 60 min of exposure. Cell viability and growth kinetics were assessed from immediate exposure to 90 min afterwards (Supplementary Figure 2). In addition, exposure to each of the environment mimicking conditions seemed to require a characteristic cellular response, as demonstrated by the principal component analysis carried out to ensure limited variation between individual samples (Supplementary Figure 3). A global and summarized overview on gene expression in transcripts per million (TPM) and DEGs (corrected p ≤ 0.05), is provided in Supplementary Table 2. DEGs were divided into upregulated (log2FC > 1) and downregulated genes (log2FC < −1), revealing characteristic transcription patterns for each sampling time point and growth condition (global overview provided in Figure 4). Based on the heatmap revealing the highest differentially expressed genes across all sample conditions investigated ( Figure 5), the most important mechanisms and pathways involved in transcriptomic responses have been further evaluated.

Global regulators
Global regulators that might be affected by the growth conditions were the class I heat shock responses comprising the HrcA regulon (hrcA-grpE-dnaK-dnaJ and groESL) that is embedded within the CtsR regulon (Michel et al., 2006) which was mostly induced, especially in the presence of ammonia (Table 1).
The center of the regulatory network in S. aureus consists of quorum sensing through the accessory gene regulator (agr)  (Bebel et al., 2020) conferring its autonomous replication capabilities. ccr, chromosomal cassette recombinase; IS, insertion sequence; DR, directs repeats associated with ccr-mediated integration and excision of SCCmec elements (Semmler et al., 2016).
Frontiers in Microbiology 07 frontiersin.org system, which concerts virulence factor transcription and synthesis, beyond others (Novick et al., 1993;Gaupp et al., 2016). We noticed a decrease in transcription of the agr locus compared to the respective unchallenged control samples for all experiments supplemented with ammonia (up to −4.7 log2FC; Table 2), a result likely related to the reduced growth kinetics associated with these conditions (Supplementary Figure 2). The addition of ammonia reduced the activity of the response regulator of the agr locus, AgrA. There was also down-regulation of AgrA-dependent genes/ operons such as the phenol soluble modulins (PSMs) encoding operon (psma and psmb), as well as of the agr-inherent effector molecule RNAIII (a larger sRNA which also includes the deltahemolysin open reading frame (hld)) noticed in ammoniachallenged cells (Supplementary Table 2). Reduced levels of RNAIII, are likely to decrease the transcription of toxin encoding genes. This is exemplified by decreased transcript levels of the α-toxin encoding gene hla (Supplementary Table 2), presumably via reduced degradation of rot transcripts, encoding the repressor of toxins (Boisset et al., 2007), which were found to be enhanced by 1.4 to 1.9 log2FC in presence of ammonia, as compared to the controls (Table 2). Reduced RNAIII levels may also explain the decreased transcript levels seen for mgrA (encoding the global transcriptional regulator MgrA) in presence of ammonia (Table 2), as RNAIII is known to stabilize the mgrA mRNA (Gupta et al., 2015).
When IMT38951 cultures were challenged with ammonia (in presence or absence of serum), clear reductions in sae (S. aureus exoprotein expression) transcript rates were noticed for both time points monitored (Table 2). Another potential factor contributing to the decrease in sae transcripts seen in presence of ammonia is SigB. This stress-induced alternative sigma factor is known to decrease sae transcription (Geiger et al., 2008), and is activated among others by a rapid increase in pH (Pané-Farré et al., 2009). Although the sigB operon is partially autoregulated, SigB activity is best followed by monitoring alkaline shock protein 23 (asp23) transcription . Here, the sigma-B regulated asp23 gene (Kuroda et al., 1995) was induced after 10 min of exposure to ammonia alone (2.4 log2FC) or the combination of ammonia and porcine serum (1.2 log2FC; Table 1). In addition, transcription of only a part of the SarA regulon was found to be altered by ammonia supplementation in a relevant manner (i.e., hla, hlgA, lrgAB, nrdG, psma, but not aur, esxA, icaADBC, nuc, sdrD, and sodM; Supplementary Table 2).

Presence of reactive oxygen species/protein synthesis
Although the equilibrium of the chemical reaction NH 3 + H 2 O ⇌ NH4 + + OH-shifts towards the left site (especially at pH 9), the rather limited number of free protons in bacterial cells (Saito and Kobayashi, 2003) may be captured by the hydroxyl radicals (OH − ). Global overview on differentially expressed genes for samples and conditions after 10 and 60 min of exposure. Pattern analysis of differentially expressed genes (DEGs) are visualized based on their log2 fold-change (log2FC) compared to the corresponding control samples, ranging from up-(yellow) to downregulated (blue). Each column corresponds to a distinct growth condition (S = serum, A = ammonia, AS = ammonia & serum) and time point (10 min and 60 min of exposure). Genes are clustered based on their respective expression across the tested conditions (left). Expression ranges from-10 log2FC to +10 log2FC.
Frontiers in Microbiology 08 frontiersin.org Heatmap of the top differentially expressed genes across all sample conditions. Top differentially expressed genes (DEGs) (n = 41) are visualized based on their respective log2 fold-change (log2FC) compared to the corresponding control samples. Each column corresponds to a stress condition (S = serum, A = ammonia, AS = ammonia & serum) and time point (10 min and 60 min). Genes are either strongly upregulated with log2FC > 5 (yellow) or downregulated with log2FC < −4 (blue). Genes are furthermore annotated with corresponding COG ontologies (left side).
Therefore, the formation of a small fraction of hydroxyl radicals (OH − ) in all samples exposed to ammonia is expected (Saito and Kobayashi, 2003). Here, we noted a transient induction of genes (maximum at 10 min of exposure) known for their response to the presence of reactive oxygen species (ROS) such as hydroxyl radicals (Pandey et al., 2019) including parts of the machinery involved in staphyloxanthin biosynthesis such as the crtOPQMN operon (Pelz et al., 2005), a carotenoid thought to protect S. aureus from oxidative stress (Clauditz et al., 2006). Similarly, genes encoding catalase (katA) and especially alkyl hydroperoxide reductase (ahpC) were most effectively induced after 10 & 60 min of exposure to ammonia alone (Table 1). Exposure to serum or even the combination of serum and ammonia did not affect transcription of the gene encoding the ribosome-associated translation inhibitor (raiA) after either 10 or 60 min, although 60 min of exposure to ammonia alone lead to a 2.0 log2FC, indicating the necessity to decrease overall protein production to cope with the presence of enhanced ammonia concentrations for more than an hour (Supplementary Table 2).

Transcriptional response of genes associated with SOS responses and horizontal transfer of SCCmec
In S. aureus, the SOS response following the occurrence of ssDNA is tightly regulated by the interplay of the DNA  Transcriptional changes after 10 min and 60 min of MRSA IMT38951 exposed to Mueller-Hinton (MH) II bouillon supplemented with 10% porcine serum, 0.3 ammonia or both, respectively. ID, sequence identifier; AA length, amino acid length. Microbiology   10 frontiersin.org Transcriptional changes after 10 min and 60 min of MRSA IMT38951 exposed to MH II supplemented with 10% porcine serum. 0.3 ammonia or both, respectively. ID, Sequence identifier; AA length, amino acid length.

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Frontiers in Microbiology 11 frontiersin.org damage-inducible repressor LexA and the recombination protein RecA (Butala et al., 2009;Maslowska et al., 2019;Podlesek and Žgur Bertok, 2020). Activation of RecA by binding to single-stranded DNA promotes transcriptional upregulation of genes belonging to the SOS-regulon by activation of LexA self-cleavage from the respective promoter sites. Although actual activation of the synthesized proteins cannot be followed by transcriptional analysis, some of the genes associated with SOS response in S. aureus are transcriptionally induced, i.e., the topoisomerase IV genes parE and parC at 10 and 60 min (1.3 /0.6 and 1.7 /0.8 log2FC) after exposure to porcine serum (Supplementary Table 2). The LexA-regulated low-fidelity and error-prone DNA polymerase V (Ha and Edwards, 2021) umuC (1.7 log2FC) and genes involved in nucleotide excision repair (uvrA/B; 1.5/1.2 log2FC) are significantly induced after 60 min of exposure to ammonia. Since a similar SOS-mediated induction of ccr genes by cleavage of LexA from its promoter sequence was reported previously , the transcriptional response of both ccr variants was evaluated. Both ccr allelic variants in IMT38951 lacked significant induction over the time points and conditions evaluated here, although a minimal induction was noted after 60 min of exposure to ammonia (between 0.1 and 0.2 log2FC; Figure 6). However, this observation seems in overall congruence with the limited but notable induction of genes belonging to the SOS-response after 60 min of exposure to ammonia described above. The transcription of lexA, on the other hand, is 0.7 log2FC after 10 and 1.5 log2FC after 60 min of exposure to ammonia, a rate that may indicate that LexA has been consumed by SOS-induction. Of note, recA transcription levels lack significant changes compared to the unchallenged controls at all conditions investigated (Supplementary Table 2). Data on up-or downregulation of predicted CDSs within the genomic region harboring SCCmec, starting with the rRNA large subunit methyltransferase (rlmH) and ending with tRNA dihydrouridine synthase (dus), is provided in Figure 6 and Supplementary Table 2 (highlighted in green). Considering genes putatively involved in mobilization and recombination of transferrable MGEs present in this particular region, we noticed an increased transcription of transposase tnp_2 associated with IS431, located adjacent to mecA, after 10 min (0.3 log2FC) and 60 min (1.0 log2FC) of exposure to ammonia ( Figure 6). Moreover, the replication initiation gene repN (10/60 min 1.0/0.5 log2FC) and the gene encoding for a plasmid recombination protein (pre; 10/60 min 1.7/0.8 log2FC), both belonging to the tetracycline resistance gene (tetK)-carrying plasmid pT181 that integrated into the SCCmec region downstream of ccrC1 ( Figure 3A), showed a transcriptional increase after exposure to porcine serum ( Figure 6; Supplementary Table 2).
Genes associated with both variants (I and II Figure 3) of the replication machinery CCPol-MP-Cch2 were not significantly induced by the growth conditions investigated here (Supplementary Table 2; Figure 6).

Discussion
Reconstruction of the loss of methicillin resistance in IMT38951_42 revealed deletion of a complete and putatively replicative SCCmecVc element by homologous recombination Following the loss of methicillin resistance in IMT38951 genomic investigations were hindered by multiple repetitive sequences in the region downstream of rlmH. This was overcome by a combination of long read sequences polished by short reads to allow for an accurate genomic inspection of this particular region, including a detailed reconstruction of the process most likely leading to the loss of the complete SCCmec element from its chromosomal integration site, outlined in Figure 3.
Previous research indicated that deletion followed by HR, or excision driven by large serine recombinases, allow complete SCCmec elements to detach from their chromosomal integration site downstream of rlmH (Hanssen and Ericson Sollid, 2006;Chlebowicz et al., 2010;Wang and Archer, 2010;Liu et al., 2017;Gómez et al., 2020). This likely requires the involvement of the serine recombinases (ccrA/B or ccrC) that facilitate chromosomal integration and excision of SCCmec, or other factors belonging to the bacterial recombination machinery. Since SCCmec elements are typically flanked by "direct repeats" (DR) of 15 bp length that are recognized by the respective large serine recombinases, ccrdriven integration or loss of these elements always leads to changes in the number of DRs at the chromosomal integration site (Ito et al., 1999;Semmler et al., 2016). However, since the number and position of the two direct repeats present in the MRSA predecessor remained unchanged in the descendent, MSSA IMT38951_42, ccr-mediated excision of the SCCmec element, as described previously (Noto et al., 2008;Wang and Archer, 2010;Zhang et al., 2015Zhang et al., , 2016, is unlikely. A further study identified recombination between the putative primase-encoding (now: helicase Cch2, according to Bebel et al., 2020) genes belonging to the CCPol-MP-Cch2 complex in the ST398 lineage (Gómez et al., 2020). Other authors have linked the loss of SCCmec with recombination between ccrC allelic variants (Chlebowicz et al., 2010;Vandendriessche et al., 2014), a possibility that cannot be ruled out for the isogenic pair belonging to ST398 in our study (Figure 3, recombination between B and C). However, the actual region prone to recombination described above comprises the end of intergenic spacer region 1 and the initial 48 bp of both ccr genes ( Figure 3A), a region that harbors four identical short repetitive sequences. Interestingly, both recombination events (A & C and B & D) events would produce an identical SCCmec circular element ( Figure 3D). Consequently, occasional deletion followed by HR might be of greater biological importance for the spread of methicillin resistance than previously considered, as Bebel et al. reported autonomous replication capabilities for exactly these elements (Bebel et al., 2020) that contribute not only to the maintenance of the circular SCCmec Frontiers in Microbiology 12 frontiersin.org intermediates, but also to their proliferation within an extrachromosomal space. Subsequently, novel SCCmec elements might be available for horizontal transfer to other suitable recipients -a hypothesis that should be addressed in future research.
In vitro transcriptional response to environmental challenges associated with pig farming highlights the adaptive capabilities of MRSA ST398 Recently, the ability of the MRSA ST398 lineage to survive in pig manure has been demonstrated (Astrup et al., 2021). This particular LA environment is known for high natural ammonia levels -a major selective factor for bacterial community structures (Nordgård et al., 2017). In S. aureus, early responses to environmental challenges are often associated with changes in the activity of global regulators (Crosby et al., 2016;Ranganathan et al., 2020;Párraga Solórzano et al., 2021). Activation of components belonging to the heat shock response seems to be among the most important coping mechanisms involved with respect to ammonia-rich environments. The latter is a protective mechanism considered crucial for bacterial survival and adaptation to hostile environmental conditions by degradation of misfolded and denatured proteins and prevention of protein misfolding events (Roncarati and Scarlato, 2017). Heatmap illustrating transcription of genes at the chromosomal SCCmec integration site from rlmH ("orfX") to dus ("orfY"). Gene transcripts are visualized based on their log2 fold-change (log2FC) with either red (upregulated), blue (downregulated) or white (no changes) compared to the corresponding control samples. Each column corresponds to a growth condition (S = serum, A = ammonia, AS = ammonia & serum) and time point (10 min and 60 min). Overall expression ranges from-2 log2FC to +2 log2FC. Microbiology  13 frontiersin.org

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The number of free protons present in a bacterial cell depends on the pH: At an alkaline pH above 9, the number of free protons is reduced to less than one per bacterium (calculated for Escherichia coli), a fact that hinders the protonation of specific sites in enzymes required for their activity (Saito and Kobayashi, 2003). Consequently, cellular stress including disturbances of pH-sensitive enzymatic reactions, energy metabolism and secondary protein structures were expected to feature in the transcriptomic responses of S. aureus cells exposed to ammonia at an alkaline pH (Gutierrez et al., 2022). Moreover, the data suggests that the electron pair of the NH 3 molecule might interfere with hybrid bonds of peptides and/or proteins under these conditions, since addition of more protein (i.e., presence of porcine serum) reduces the effects associated with exposure to ammonia alone, arguing against a pH dependent effect alone. In line with this, the initial pHs of the ammonia-supplemented samples differed only slightly (ammonia: pH 9.0/ammonia & porcine serum: pH 8.9) and did not change significantly over time.
A reduction of the bacterial transcription machinery prevents excessive energy loss from protein synthesis (Roncarati and Scarlato, 2017). As shown in Supplementary Figure 2, viable cell numbers had the smallest increase over time in samples exposed to ammonia only, suggesting that down-regulation of the translation machinery (Supplementary Table 2) contributes to survival and adaptation in ammonia rich environments.
In addition, the asp23 expression that is controlled by the alternate sigma factor of RNA polymerase (SigB) was upregulated after 10 min of exposure to 0.3% ammonia and the combination of ammonia and porcine serum, an increase that has been reported for sodium hydroxide-induced pH increases previously (Anderson et al., 2010). However, after 60 min, the asp23 transcription rates were comparable to the control, suggesting that ammonia supplementation induces a short-lived activation of SigB. This is in line with earlier observations reporting that SigB activity is only transiently induced by stresses such as heat shock (Gertz et al., 1999;Giachino et al., 2001). Further support for a stimulating effect of ammonia on SigB activity is given by our findings of increased transcript rates for several SigB-regulated genes known to be directly controlled by this factor such as the genes crtMN, whose products are involved in staphyloxanthin biosynthesis, a carotenoid thought to protect S. aureus from oxidative stress (Clauditz et al., 2006), spoVG, a transcription factor acting downstream of SigB (Meier et al., 2007), and sarA . Notably, SarA is also reported to act as regulatory protein responsive to redox and pH (Fujimoto et al., 2009), and to repress the expression of rot (Manna and Ray, 2007).
The observed reduction in sae transcription in all samples except those exposed to porcine serum for at least 60 min might be, at least in part, due to increases in SigB and Rot-activities, which were shown to decrease sae transcription from the sae P1 and P3 promoters, respectively (Geiger et al., 2008;Li and Cheung, 2008), and by a reduction in RNAIII transcripts, which are known to enhance sae transcription (Novick and Jiang, 2003;Geiger et al., 2008).
Although not directly regulated by agr, gene transcription of a few virulence factors located on mobile genetic elements that can be considered as a specific response to host (porcine) proteins such as scn and the von Willebrand Factor binding protein were decreased in all samples exposed to ammonia with or without porcine serum, indicating that the pH of 8.9-9.0 and/or the NH 3 molecule interferes with (host-)protein recognition, requires energy saving or both.
Transcription of genes involved in capsular polysaccharide biosynthesis in ammonia supplemented media was not recorded, although their upregulation due to increased alkaline conditions has been demonstrated before (Anderson et al., 2010).
Taken together, LA-MRSA ST398 seems fully equipped to endure pig-farm associated environmental factors such as ammonia and porcine serum, helping to support their long-term survival within that environment.

Transcription of genes involved in SCCmec mobility
In S. aureus, the genomic region between origin-of-replication (ori) and the SCC generally seems prone to HR, with the integration site for SCC representing a particular "hotspot" in this regard (Everitt et al., 2014;Semmler et al., 2016). A recent study investigated the stability of four different SCCmec elements (I, II, III, IV) during 3 months of serial subculturing at room temperature, revealing that SCCmec stability was influenced both by internal mobile elements (IS431) as well as the cell environment (Scharn et al., 2022). Exposure to pig-farming associated environmental challenges did not significantly alter the expression ratios of genes likely to be involved in the mobilization of complete or partial SCCmec elements, including transposases associated with IS431 and the ccr recombinases (Wang and Archer, 2010;Wang et al., 2017;Scharn et al., 2022) in this study when compared to unchallenged controls. These results suggest that the aforementioned conditions do not strongly contribute to SCCmec mobility to a large extent -at least not within the timeframe investigated here.
Moreover, exposure to ammonia, porcine serum or both failed to significantly induce recA transcription that would lead to de-repression of LexA-controlled genes including allelic variants of ccrC, as reported before . Although some genes tightly regulated by LexA such as uvrAB and umuC showed a clear log2FC increase after 60 min of exposure, the corresponding transcription rate noted for both ccr genes (0.1/0.2 log2FC) lacked significant difference compared to the controls. Since autocatalysis of LexA is required for derepression of some genes within the SOS regulon (Ha and Edwards, 2021) including lexA, others are only derepressed when the N-terminal domain is further digested by Clp proteases including ClpCP. Here we noted an induction between 2.5 log2FC after 10 min and 4.9 log2FC after 60 min for clpC in cells exposed to ammonia only (Table 1), once more suggesting a fine-tuned activation of the SOS machinery in MRSA ST398 strain IMT38951. Therefore, a massive cleavage of LexA from the ccrC promoter region may depend on more invasive DNA Frontiers in Microbiology 14 frontiersin.org damaging conditions, i.e., the presence of distinct antibiotics, as reported before (Cirz et al., 2007;Liu et al., 2017). It seems worth considering that DNA-damaging effects attributed to ammonia rich environments and further challenging circumstances, for instance, exposure to antibiotics, may accumulate in vivo. In light of the recent results of Scharn et al. (2022), we speculate that prolonged exposure to livestock-associated conditions might induce a more prominent transcription of the ccr genes.

Conclusion
Here we followed the deletion of a complete and fully functional SCCmec Vc element from its chromosomal integration site downstream of rlmH in an MRSA ST398 isolate (IMT38951) and highlighted the most important transcriptional responses of the bacteria required to endure pig-farm associated environmental challenges such as increased ammonia concentrations and porcine serum. A detailed reconstruction of SCCmec loss from its genomic integration site raises concerns with respect to the autonomous replication capabilities of the predicted circular SCCmec element and its potential to spread to other susceptible bacterial hosts. It seems likely that this particular method of SCCmec conservation and transmission might have been underestimated, especially considering that, at least so far, only severe DNA-damaging events seem to trigger an increased activity of the ccrC variants at the population level. However, further studies are required to evaluate which factors and co-factors may trigger and support the autonomous replication machinery present in the excised elements.

Data availability statement
The datasets presented in this study can be found in online repositories. Raw sequencing reads, including RNA-Seq and longread genomic data, were uploaded to NCBI and deposited within BioProjects PRJNA891722 and PRJNA449454.

Author contributions
CH, AL-B, LW, LE, SW, and BW contributed to conception and design of the study. CH, JB, JA, and BW performed laboratory experiments and analysis. Genomics and transcriptomics were carried out by LE, SW, AT, and TS. CH, MB, MH, AB, WZ, and BW analysed the results. CH and BW wrote the first draft of the manuscript. LE, SW, WZ, and MB wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.

Funding
This work was supported by the German Federal Ministry of Education and Research (BMBF) for #1Health-PREVENT (grant nos. 01KI2009D and 01KI2009F) and PAC-CAMPY (grant no. 01KI2007F) within the German Research Network of Zoonotic Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CH received a research doctoral fellowship from Akademie für Tiergesundheit e.V. (AfT; Germany).


Short Communication: Use of Infrared Thermometers for Cutaneous Temperature Recording: Agreement with the Rectal Temperature in Felis catus

Simple Summary The recording of body temperature by rectal temperature assessments is a stressful procedure for cats. For this purpose, alternative methods for using rectal digital thermometers to monitor body temperature were investigated. Skin temperature was recorded in 20 cats, in 5 different body regions, and compared with the rectal temperature. The obtained data indicated that the cutaneous temperature recorded by the infrared thermometers was not in agreement with the data recorded by the digital thermometer in the rectum. Abstract In veterinary medicine, the gold standard for assessing body temperature is rectal temperature assessment. Considering that this procedure is stressful for many species, in particular for cats, it could be clinically important to consider an alternative approach for the monitoring of core body temperature. The aim of this study was to test if cutaneous temperature measurements by means of different infrared thermometers are in agreement with the most commonly used method for body temperature measurement in cats. The cutaneous temperature was recorded in the jugular, shoulder, rib, flank, and inner thigh, using three different non-contact infrared thermometers (IR1, IR2, and IR3) in 20 cats. The cutaneous temperature was then compared to the rectal temperature, recorded by means of a digital thermometer. The obtained data indicated that the cutaneous temperature recorded by the infrared thermometers was not in agreement with the data recorded by the digital thermometer in the rectum. In cats, the use of non-contact infrared thermometers gave no reproducible or constant data to justify their application for the recording of body temperature instead of rectal temperature recording. In addition, the infrared temperature measurement devices generated results that were not in good agreement among themselves, providing a novel result of clinical importance.

Introduction
Temperature measurement is an important step in clinical examinations of animals because there is an evident association between temperature and disease [1], and it represents a valuable tool for monitoring the physiological status, welfare, and stress responses of animals [2]. The core body temperature is assessed by an invasive contact device, such as an esophageal or a pulmonary artery thermistor, although these are not suitable for conscious veterinary patients [3,4]. In veterinary medicine, rectal temperature measurement has traditionally been the most common method for assessing body temperature. However, obtaining the rectal temperature can be a stressful procedure for patients, particularly if serial measurements are needed [5,6]. Some authors showed that rectal temperature measurement could provoke defensive behaviors from the animal under examination [7]. This invasive procedure, in reactive and stressful animals, might alter the result of the measurement, thus compromising its interpretation and the consequent diagnostic and therapeutic measures [8]. In fact, anxiety and stress affect many clinical measurements used to evaluate animal health [9][10][11][12]. In particular, in homeothermic animals, it has been documented that acute and chronic stress are associated with a rise in the core body temperature [13]. In clinical environments, cats can experience considerable anxiety and stress because of unfamiliar surroundings, people, and methods of handling. Furthermore, the effects of stress on cats may persist far beyond the clinic visit. Evidence-based best-practice guidelines recommend low-stress handling procedures and reducing the use of invasive practices, replacing them with non-restrictive practices [14,15]. Modern measurement techniques are based on the use of diagnostic tools that reduce a subject's stress by a quick assessment of the body temperature, without the need for restraint or contact [16].
In human medicine, body temperature measurement methods comprise conductive thermocouples, thermistors, and telemetry systems, as well as contact-free infrared thermometry and imaging [17,18]. Infrared (IR) methodologies determine the surface body temperature by measuring the natural electromagnetic radiation emitted from the body [2]. Several infrared thermometers have been developed specifically for veterinary use, resulting in better patient compliance and quicker results. The body temperature is highly variable and is influenced by a vast range of environmental, physiological, and pathological factors, including ambient humidity and temperature, and disease processes; indeed, the core body temperature brings about changes in the body temperature [2,[5][6][7][8]18,19]. Thermal comfort is guaranteed by the transfer of heat by the cutaneous vasomotor adjustments that permit or reduce the convection between the body's core and the skin surface through the bloodstream [20]. Skin temperature changes have also been associated with the vasoconstriction and vasodilatation of microcirculation due to an autonomic nervous system response to inflammation, infectious, neoplastic, pain-induced, or stress effects [19].
In human medicine, the measurement of the body temperature of a specific skin region-for example, the axillary temperature-seems to be the least stressful method for assessing temperature because of the minimal cooperation required, not only in children [21]. The surface body temperature can be determined using thermal windows, referring to regions with surface blood vessels where changes can be detected that represent surface circulation. Infrared thermography is used for the diagnosis of and follow-ups for several disorders, including vascular, orthopedic, neurologic, and neoplastic diseases that cause changes in the surface body temperature. Different skin regions were proposed in cats to assess superficial temperature [5,19], such as ocular, abdominal [22], dorsal and lateral views of the entire body and paw print to assess painful conditions [23], and pelvic limbs to detect aortic thromboembolism [24].
In adult and pediatric practice, an inconsistent correlation of infrared methodology with core or rectal temperatures was reported [25][26][27].
In dogs, the auricular temperature measured with an infrared thermometer was similar to the body temperature obtained with a rectal thermometer [41][42][43], also during the monitoring of hyperthermia induced by physical exercise [38]. The gum temperature exhibited the best clinical potential when digit, snout, axilla, eye, gum, inguinal region, and anal verge temperatures were compared to rectal temperature [44].
In cats, many studies have been conducted to validate the use of alternative methods for recording the body temperature and rectal temperature. The discussion of the clinical use of contact-free infrared thermometry has centered on various anatomical regions that could be used as sites for cutaneous temperature measurements, such as ear, pinna, preauricular area, tympanic membrane, nasal planum, gingival mucosa, eye, dorsal neck, lateral neck, ventral neck, thorax, axilla, metacarpal pad, abdomen, medial thigh, lateral thigh, tail, and perineum [5][6][7][45][46][47][48]. A marked surface temperature variation was shown in cats by a forward-looking infrared camera. Therefore, numerous studies compared the reliability and repeatability of skin temperature measurements at different sites [5].
Ocular temperature has been considered an alternative method for body temperature measurement. When ocular temperature is used, it is necessary to consider that the surface temperature of the eye is 1.2 • C less than the rectal temperature in cats [40]. In other body regions, the obtained results are discordant: some authors indicated the use of the auricular thermometer as a reliable alternative to rectal thermometry for the assessment of body temperature [43], while others reported that the axillary and tympanic membrane temperatures were not interchangeable with the rectal temperature [46].
Despite the large existing literature on infrared thermometry, a reference range for diverse body regions' temperature measurements in cats needs to be validated. Moreover, there is an evident gap in the existing literature regarding the comparison of different IR thermometer models for measuring surface body temperature in cats, in order to verify their interchangeability.
The aim of this study was to evaluate the use of non-contact infrared thermometers in healthy cats by the investigation of five body regions and by the comparison of three different non-contact infrared thermometers. The objective of the study was to verify if the cutaneous temperature measurement by means of different infrared thermometers is in agreement with rectal temperature, identifying which body regions and infrared devices could be more appropriate for this scope.

Animal and Experimental Design
Twenty client-owned cats of different breeds (Maine Coon, European Shorthair, and Ragdoll), 10 females and 10 neutered males, 3-5 years old, with a mean body weight 4.5 ± 0.20 kg, with a coat color from white to grey, and with coat lengths between shortand medium-hair were recruited for the study. Cats examined between March and June 2020 were eligible for inclusion. Cats suffering from ectoparasites, recto-anal disease, dermatitis, cutaneous neoplasia, and metabolic and contagious diseases were excluded, as were any cats that had a rectal temperature outside the range of 37.8 • C to 39.4 • C, classified as normothermia [41,46]. This work involved the use of non-experimental animals only (owned animals). The established, internationally recognized high standards ("best practice") of individual veterinary clinical patient care were followed. The protocol of animal experimentation was reviewed and approved by the Ethics Review Board, in accordance with the standards recommended by the Guide for the Care and Use of Laboratory Animals and Directive 2010/63/EU for animal experiments. All animals were enrolled in the study after receiving the written consent of the owners, in compliance with the Italian Regulation D.L. 116/1992.

Measurements
Cutaneous temperatures were recorded by means of three different non-contact infrared thermometers in five fully haired regions of interest: jugular, shoulder, rib, flank, and inner thigh ( Figure 1). The measurement sites were standardized on the right side using anatomical landmarks as follows: jugular site at the middle-third of the jugular vein; shoulder site at the tip of the shoulder; rib site at the middle-third of the chest, caudally to the heart area; flank site at the dorsal portion of the flank, cranially to the iliac crest; and inner thigh site, caudally to the knee joint. The mentioned five regions of interest were chosen because animal handling is not required for reading the surface temperature in those areas [5].
The infrared thermometers used do not have a conversion factor incorporated, which converts a cutaneous reading into an estimated equivalent rectal reading.
The temperature and humidity conditions inside the experimental room were constantly monitored using a thermohygrometric probe. The microclimatic conditions were evaluated (mean ± standard deviation (SD)): the environment temperature was 20 ± 0.6 • C and the humidity was 47 ± 1.5%.
Prior to the use of infrared thermometers, the subjects were kept in the experimental room for approximately 15 min for acclimatization to the environmental conditions. Three different infrared thermometers were used to assess the cutaneous temperature: IR1, the infrared thermometer Norditalia FI-150, with an accuracy of ±1.0 • C; IR2, the infrared thermometer THM010-VT001, Mediaid Inc., Cerritos, CA, USA, with an accuracy of ±0.3 • C; and IR3, the Fluke 62 mini-infrared thermometer, with an accuracy of ±1.5 • C. All the thermometers were placed at a distance of 5 cm from the measurement site. They were equipped with a tube placed above the sensor to reduce the sensor's field of view (FOV) and to remove possible ambient IR sources. In total, 16 measurements were made for each cat. A single rectal temperature was measured using a digital thermometer (model HI92704, Hanna Instruments, Bedfordshire, UK), with a resolution of 0.1 ºC, that was inserted 3 cm into the rectum.
The order of data recordings by means of the IR thermometers and the measurement sites were chosen randomly. The rectal temperature was evaluated after the cutaneous temperature to avoid false results because of temperature elevations due to activity and muscular exertion, resulting in a local temperature increase in the rectum [47].
All the cats enrolled in the study were accustomed to human presence and contact. During the experimental protocol, none of the cats showed behavioral disorders related to acute stress induced by cutaneous temperature measurement [32]. The same operator performed the temperature recordings.
To assess the accuracy of the digital and IR thermometers, we used them to evaluate the thermal recovery of an object preheated inside a thermostated oven, and then left it at room temperature for 15 min. A breast phantom, usually employed for quality controls of radiological equipment and made of material mimicking human tissue, was used.
All the temperature recordings were performed at the same time of the day (09:00) to avoid changes due to the circadian oscillation of the body temperature, as observed in diurnal animals [32]. Owned cats have been observed to show diurnal activity, with a higher amount of activity during the photophase than during the scotophase, as a consequence of the close relationship with humans, inducing an adaptation of their lifestyle to those of their owners [33].

Statistical Analysis
A Kruskal-Wallis test was applied to evaluate the consistency of the experimental data recorded during the evaluation of the preheated object for the 15 min of the thermal recovery.
The obtained data, expressed as mean ± SD, were normally distributed (Kolmogorov-Smirnov test, p > 0.05). Two-way analysis of variance (ANOVA) was applied to determine statistically significant effects of the thermometer and body region on the cutaneous temperature measured in cats. Tukey's test was applied for post hoc comparison. For the comparison among the different IR devices and body temperature measurements, the percentage of paired readings, in which the rectal-body temperature differences were within the clinically accepted limit of ±0.5 • C, was determined.
The correlation coefficient (r) between rectal temperature and the cutaneous temperature recorded with the means of the various used thermometers in each tested region was evaluated. Agreement between the body temperature measurements and the rectal temperature measurements, and among the different thermometers, were determined by the Bland-Altman method. Bias was defined as the mean difference between two methods, and the limits of agreement (LOA) were calculated as the bias ± 1.96 (SD), as previously reported [4,5,27]. A value of p <0.05 was considered statistically significant. The data were analyzed using the statistical software Prism v.5.00 (Graphpad Software Ltd., San Diego, CA, USA, 2003).

Results
The results of the measurements of an object preheated inside a thermostated oven and then left at room temperature for 15 min are presented in Table 1. The application of the Kruskal-Wallis test indicated no significant differences between the measurements obtained from the four different instruments (p = 0.98542, significance level p < 0.01). Having assessed the consistency of the probes, we proceeded with the experimental protocol on cats. Table 1. Temperature ( • C) recorded for the accuracy assessment of the different thermometers used during the experimental protocol. The temperature of a preheated object was recorded for 15 min during its thermal recovery every 1 min by means of digital and infrared (IR) thermometers.  Table 2 reports the mean ± SD of the cutaneous temperature recorded in the five regions by means of the three different non-contact infrared thermometers and of rectal temperature recorded by a digital thermometer. The rectal temperature recorded in the 20 healthy cats was 38.59 ± 0.60 • C. The two-way ANOVA showed a significant effect of the different thermometers (p < 0.0001) and body regions (p < 0.0001) on the cutaneous temperatures in the cats. In particular, regarding the data recorded by the different thermometers, IR3 recorded a lower temperature than IR1 in all the body regions investigated (jugular p < 0.0001, shoulder p < 0.0001, rib p < 0.0001, flank p < 0.05, inner thigh p < 0.0001). IR3 recorded a lower temperature than IR2 in the jugular (p < 0.01) and shoulder (p < 0.01) regions. IR2 recorded a lower temperature than IR1 in the jugular (p < 0.0001), shoulder (p < 0.05), rib (p < 0.0001), and inner thigh (p < 0.05) regions. Regarding the various body regions investigated, IR1 gave a higher temperature in the inner thigh than in the jugular, rib, and flank (p < 0.01). IR3 recorded a lower value of the cutaneous temperature in the jugular region than in the shoulder (p < 0.01), rib (p < 0.01), flank (p < 0.0001), and inner thigh (p < 0.0001) regions; moreover, the temperature of the inner thigh region was higher than that in the shoulder region (p < 0.01). The use of IR2 did not show differences in the recording of the cutaneous temperature in the various regions studied. Table 2. Means ± SD of cutaneous temperature recorded in the five body regions (jugular, shoulder, rib, flank, inner thigh) obtained with the three different thermometers, and rectal temperature recorded by a digital thermometer in twenty healthy cats, expressed in • C. Statistical significance is also reported. Symbols ( # = vs. jugular region, * = vs. shoulder region, • = vs. rib and flank) indicate statistically differences of cutaneous temperature between the five different body regions (jugular, shoulder, rib, flank, inner thigh) using the same thermometer. Lower letters ( a = vs. IR1 and IR2, b = vs. IR1) indicate statistically different cutaneous temperature values recorded in the same cutaneous region using different thermometers (IR1, IR2, IR3). The mean cutaneous temperature values from IR1, IR2, and IR3 tended to be lower than the rectal temperature by, respectively, 1.38 • C to 3.04 • C, 3.42 • C to 4.18 • C, and 4.51 • C to 7.08 • C. Out of 300 measurements, 3.43% of cutaneous temperature measurements by the three IR thermometers within ±0.5 • C of rectal temperature measurements was reported.

Thermometers
The rectal temperature value recorded by means of digital thermometer was positively correlated with the cutaneous temperature recorded at the jugular (p < 0.05; r = 0.61), shoulder (p < 0.05; r = 0.66), and flank (p < 0.01; r = 0.75) regions with the IR1 thermometer.
Agreement between the cutaneous and rectal temperature measurements was not satisfactory, as shown by the Bland-Altman plots (Figure 2), in which the mean temperature values were far from the zero-bias line, with a large SD of the bias and a large width of the limits of agreement. In cutaneous infrared measurements, the temperature was overestimated at lower values of the rectal temperature range, and underestimated at higher values.
Considering the comparison of the various IR thermometers used, with respect to the rectal one, the lowest bias was observed using the IR1 in all investigated regions; considering the various investigated body regions, the lowest bias was observed in the inner thigh (Table 3).

Figure 2.
Bland-Altman comparisons of the rectal temperature values recorded with a digital thermometer, with the cutaneous temperature recorded using the three different non-contact infrared thermometers (IR1, IR2, and IR3) on the different cutaneous sites (jugular, shoulder, rib, flank, and inner thigh) in twenty healthy cats. The dotted lines represent the lower (L) and upper (U) limits of agreement (LOA); the solid line represents the mean difference (bias: B rectal temperature-cutaneous temperature) between the cutaneous and the rectal temperature measurements. Table 3. Bland-Altman comparisons of the cutaneous temperature recorded (the jugular, shoulder, rib, flank, and inner thigh) with three different non-contact infrared thermometers (IR1, IR2, and IR3) in twenty healthy cats.

Discussion
In the current study, we compared three non-contact infrared thermometers with a digital rectal thermometer in the evaluation of body temperature in healthy cats by the investigation of five body regions. As expected, the comparison of the rectal and cutaneous temperatures showed a higher rectal temperature value than the cutaneous one. Although measurements of jugular, shoulder, and flank cutaneous temperature by only one IR thermometer correlated modestly with rectal temperatures, the agreement between the two methods was too poor to recommend the use of the tested infrared devices in clinical practice.
The obtained results are in accordance with previous studies conducted with different strategies and infrared thermometers, and in different animal species [2,44].
Kunkle et al. [45] reported highly variable values using an infrared thermometer for the body temperature measurements (a mean difference of 0.07 • C and limits of agreement of 1.43 • C and −1.36 • C), which they considered unacceptable for clinical purposes in cats.
According to the present results, it is not only the differences between the rectal and cutaneous temperature that make the use of infrared thermometers unacceptable for the monitoring of body temperature in cats, but also the fact that the infrared devices generated results that were not in good agreement among themselves. The obtained data were not reproducible and not constant.
To date, despite the large existing literature on infrared methodology in veterinary medicine [2][3][4][5][6][7][8]16,18,19,[22][23][24], no attention has been given to monitoring the performance of different infrared devices, comparing their surface temperature measurements in cats. In other species, the differences between rectal and cutaneous temperature have been established to be a conversion factor when the alternative method is used for the recording of body temperature instead of the gold standard of rectal temperature [29]. The infrared thermometers used do not have a conversion factor incorporated, which converts a cutaneous reading into an estimated equivalent rectal reading, strengthening the explanation that the detected differences among the various cutaneous sites and rectal temperature measurements were due to physiological temperature differences.
The comparison of the cutaneous temperature measurements on the different body regions and by the three examined thermometers showed that the IR3 thermometer recorded lower mean cutaneous temperature values at the jugular region than at the shoulder, flank, rib, and inner thigh regions.
The measurement values obtained with IR2 showed no statistically significant differences among them. IR1 recorded higher temperature values than all the cutaneous thermometers used in this study. The jugular, shoulder, and inner thigh showed different temperature values using all tools, whereas the rib and flank provided different values with IR3 as compared with IR1.
The differences observed among the recorded data may represent a physiological difference between body sites. The differences in the cutaneous temperature could be due to the relative power of the different body regions to emit heat by radiation (emissivity), linked to the length, color, and density of the coat, the thinner fur, and the muscle mass [36,37]. It is likely that the hair coat impeded infrared emission detection at some sites, which would explain why lower temperature readings were associated with a greater length of the coat [5,52]. The different arterial blood flows in each body portion represent another factor that can play a role in the variability of the data [5]. The same expert operator performed the temperature measurements, thus excluding the inter-rater variability.
The assessed regions showed different cutaneous temperature values, also regarding the infrared methodology used. The three IR methodologies showed low repeatability of data recordings in vivo, in contrast to the high repeatability observed during the test performed on the heated object before the start of the study. This finding is in agreement with previously reported findings in feline species regarding the use of tympanic infrared thermometers [7]. However, the reason for this difference is not clear and needs further investigation.
In an ideal instrument, the difference between infrared and rectal thermometry would be 0 • C at any temperature measured [27]. Statistical analysis showed poor agreement among the cutaneous thermometry data with wide limits of agreement and large bias, confirming that cutaneous temperature measurement in the studied body regions cannot be used as a reliable substitute for rectal temperature measurement in clinical settings. Moreover, the fact that less than 4% of cutaneous temperature measurements in different body sites and with different devices were not within ±0.5 • C of rectal temperatures suggests that these methods should not be used interchangeably in cats. The poor interchangeability is also due to the fact that the infrared devices generated results not in good agreement with them, despite their similar technical characteristics.
In addition to the only moderate correlation between methods, all of the cutaneousrectal temperature Bland-Altman plots showed variable bias, with generally positive temperature differences at low average body temperatures, and generally negative temperature differences at high average body temperatures, suggesting perhaps that surface temperature remains relatively constant despite changes in rectal temperature. This finding further underlines how the cutaneous temperature differs from the rectal one.
Our results are striking in that we observed this low concordance between rectal and infrared temperatures in healthy cats maintained in a zone of thermal neutrality at standardized external conditions to reduce the effects of external variables. Infrared temperature can be expected to state more than 1 • C (until about 7 • C) below a rectal temperature, and this discrepancy precludes the use of infrared thermometry in feline practice.
As reported in hypo-and hyperthermic cats [5] and in young febrile children [26], this study showed a significant device overestimation of infrared temperature at low rectal temperatures and an underestimation at elevated rectal temperatures also in normothermic cats.
The present study has some limitations, such as the small sample size and the lack of hypothermic and hyperthermic measurements to permit sensitivity/specificity testing to detect abnormal body temperatures. Although the small sample size may be warranted by the narrow inclusion criteria necessary to obtain a homogeneous sample of healthy cats, further studies are needed on a larger number of cats to confirm our results in different environmental conditions and in hypo/hyperthermic subjects.

Conclusions
This study demonstrated that infrared temperature measurement devices generated results that were not in good agreement among themselves, providing a novel result of clinical importance. In the present study, infrared thermometry used in the region of interest of the jugular, shoulder, rib, flank, and inner thigh does not correlate to rectal temperature. So, the cutaneous temperature does not seem to be a reliable alternative to the rectal temperature for measuring the body temperature in cats. In cats, the use of non-contact infrared thermometers gave no reproducible or constant data to justify their application for the recording of body temperature instead of rectal temperature recording. In addition, the absence of reproducible data prevented the establishment of the normal cutaneous temperature range in the various body regions investigated.
Author Contributions: Conceptualization, C.G. and S.D.P.; formal analysis, F.A.; investigation, A.F. and E.G.; resources, G.P.; data curation, F.A.; writing-original draft preparation, G.A. and M.P.; writing-review and editing, C.G., supervision S.D.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Institutional Review Board Statement:
This work involved the use of non-experimental animals only (owned animals and data from prospective or retrospective studies). Established internationally recognized high standards ("best practice") of individual veterinary clinical patient care were followed. Informed Consent Statement: Written informed consent was obtained from the owners of all animals described in this work for the procedures undertaken. For any animals or humans individually identifiable within this publication, informed consent for their use in the publication (verbal or written) was obtained from the people involved.

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.